A greatly expanded thermodynamic database for modified oligonucleotides will be developed in this project and incorporated into existing design software. This Fast Track SBIR proposal focuses on determining thermodynamic parameters for modifications that are currently finding widespread applications. The new thermodynamic database and software will enable a large number of researchers throughout the world to more easily develop advanced applications with modified oligonucleotides and will add substantial commercial value to the software products. In this new proposal, the range of modified oligonucleotides will be greatly increased to include a variety of non-nucleotide linkers and 13 additional modified nucleotides that have found the widest applications. These modifications include modified nucleotides that improve thermodynamic stability (2,6- diaminopurine, 2'-O-methyl RNA, 5-propynylpyrimidines), universal bases (5-nitroindole and 3-nitropyrole), modified oligonucleotides used in therapeutics (phosphorothioates, 2'-O-methyl RNA, 2'-fluoropyrimidines) and modified oligonucleotides with other applications (2-aminopurine, 2-thiothymine, 5-methylcytosine, 7- deazaguanine). This work complements the work done on other modified nucleotides whose thermodynamic parameters were measured during a previous project (under R44GM076745). The study involves a systematic series of measurements on over 500 modified duplexes along with unmodified controls. The newly determined parameters will be incorporated into a software database to enable simulation and in silico optimization of hybridization experiments in the presence of a wider coverage of modified oligonucleotides than previously possible. This will aid researchers in designing more robust assays and therapeutics. Modified oligonucleotides can be categorized either by the structure of the modified nucleotide or by its intended application. In this proposed research, a representative sample of the most commonly used modifications, both by structure and by function, is examined. Although many modifications are used in combination with one another, it is not proposed to study combinations of different modifications within the same oligonucleotide in this project as the number of possible combinations would make this task untenable. Some examples of modifications used in combination include: 2'-O-methyl-RNA containing terminal phosphorothioate, LNA-2'-O-methyl-RNA combinations, and phosphorothioate-LNA combinations. In addition to the measurements of thermodynamic parameters for oligonucleotides not previously measured, it will be necessary to restructure the software modeling and simulation program to allow for the inclusion of these nucleotides in both calculation and display modules of the program.